• Asian Pacific Journal of Cancer Prevention
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• v.14 no.10
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• pp.5825-5831
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• 2013

Background/Objectives: Maintenance of cellular function in culture is vital for transfer and development following adoptive immunotherapy. Dual properties of IL-21 in activating T cells and reducing activation induced cell death led us to explore the mechanism of action of IL-21 enhanced proliferation and cytotoxic potential of CIK cells. Method: CIK cells cultured from PBMCs of healthy subjects were stimulated with IL-21 and cellular viability and cytotoxicity to K562 cells were measured. To elucidate the mechanism of action of IL-21, mRNA expression of cytotoxic factors was assessed by RT-PCR and protein expression of significantly important cytotoxic factors and cytokine secretion were determined through flow cytometry and ELISA. Western blotting was performed to check the involvement of the JAK/STAT pathway following stimulation. Results: We found that IL-21 did not enhance in vitro proliferation of CIK cells, but did increase the number of cells expressing the CD3+/CD56+ phenotype. Cytotoxic potential was increased with corresponding increase in perforin ($0.9831{\pm}0.1265$ to $0.7592{\pm}0.1457$), granzyme B ($0.4084{\pm}0.1589$ to $0.7319{\pm}0.1639$) and FasL ($0.4015{\pm}0.2842$ to $0.7381{\pm}0.2568$). Interferon gamma and TNF-alpha were noted to increase ($25.8{\pm}6.1ng/L$ to $56.0{\pm}2.3ng/L$; and $5.64{\pm}0.61{\mu}g/L$ to $15.14{\pm}0.93{\mu}g/L$, respectively) while no significant differences were observed in the expression of granzyme A, TNF-alpha and NKG2D, and NKG2D. We further affirmed that IL-21 signals through the STAT-3 and STAT-5b signaling pathway in the CIK cell pool. Conclusion: IL-21 enhances cytotoxic potential of CIK cells through increasing expression of perforin, granzyme B, IFN-gamma and TNF-alpha. The effect is brought about by the activation of STAT-3 and STAT-5b proteins.

• Biomedical Science Letters
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• v.13 no.3
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• pp.197-206
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• 2007

P2X receptors are membrane-bound ion channels that conduct $Na^+,\;K^+$, and $Ca^{2+}$ in response to ATP and its analogs. There are seven subunits identified so far ($P2X_1-P2X_7$). $P2X_2$ receptors are known to be expressed in a wide range of organs including brains and adrenal grands. PC12 cells are originated from adrenal grand and differentiated by nerve growth factor or pituitary adenylate cyclase activating poly peptide (PACAP). Previous studies indicate that $P2X_2$ receptor activation in PC12 cells couples to $Ca^{2+}-dependent$ release of catecholamine and ATP. It is known that acidic pH potentiates ATP currents at $P2X_2$ receptors. This leads to a hypothesis that $P2X_2$ receptors may play an important role in PC12 cell differentiation, one of the characteristics of which is neurite outgrowth, induced by the hormones under lower pH. In the present study, we isolated several clones which potentiate neurite outgrowth by PACAP in acidic pH (6.8), but not in alkaline pH (7.6). RT-PCR and electrophysiology data indicate that these clones express only functional $P2X_2$ receptors in the absence or presence of PACAP for 3 days. Potentiation of neurite outgrowth resulted from PACAP (100 nM) in acidic pH is inhibited by the two P2X receptor antagonists, suramin and PPADS ($100\;{\mu}M)$ each), and exogenous exprerssion of ATP-binding mutant $P2X_2$ receptor subunit ($P2X_2[K69A]$). However, acid sensing ion channels (ASICs) are not involved in PACAP-induced neurite outgrowth potentiation in lower pH since treatments of an inhibitor of ASICs, amyloride ($10\;{\mu}M$), did not give any effects to neurite extension. The vesicular proton pump ($H^+-ATPase$) inhibitor, bafilomycin (100 nM), reduced neurite extension indicating that ATP release resulted from $P2X_2$ receptor activation in PC12 cells is needed for neurite outgrowth. These were confirmed by activation of mitogen activated protein kinases, such as ERKs and p38. These results suggest roles of ATP and $P2X_2$ receptors in hormone-induced cell differentiation or neuronal synaptogenesis in local acidic environments.

• BMB Reports
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• v.41 no.2
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• pp.112-118
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• 2008

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 ${\mu}M$ methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1637-1642
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• 2015

Background: RhoGTPase-activating proteins (RhoGAPs) regulate RhoGTPases in cells, but whether individual reactive oxygen species (ROS) regulate RhoGAPs is unknown. Our previous published papers have shown that deleted in liver cancer 1 (DLC1) inhibits cancer cell migration by its RhoGAP activity. The present study was designed to explore the role of $H_2O_2$ in regulation of DLC1. Materials and Methods: We treated cells with $H_2O_2$ for 24h and phenotypic changes were analyzed by MTT, RT-PCR, Western blotting, immunofluorescence staining and wound healing assays. Results: $H_2O_2$ downregulated cyclin D1 and cyclin E to inhibit proliferation, and upregulated BAX to induce apoptosis in MCF-7 cells. Compared with non-tumorigenic cells, $H_2O_2$ increased expression of DLC1 and reduced activity of RhoA in cancer cells. Stress fiber production and migration were also suppressed by $H_2O_2$ in MDA-MB-231 cells. Conclusions: Our study suggests that $H_2O_2$ inhibits proliferation through modulation of cell cycle and apoptosis-related genes, and inhibits migration by decreasing stress fibers via DLC1/RhoA signaling.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.1901-1906
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• 2012

Background: The discovery that microRNA (miRNA) regulates metastasis provide a principal molecular basis for tumor heterogeneity. A characteristic of solid tumors is their heterogenous distribution of blood vessels, with significant hypoxia occurring in regions (centers of tumor) of low blood flow. It is necessary to discover the mechanism of breast cancer metastasis in relation to the fact that there is a differential distribution of crucial microRNA in tumors from centers to edges. Methods: Breast tissues from 48 patients (32 patients with breast cancer) were classified into the high invasive and metastatic group (HIMG), low invasive and metastatic group (LIMG), and normal group. Samples were collected from both the centers and edges of all tumors. The first six specimens were detected by microRNA array, and the second ten specimens were detected by real-time qRT-PCR and Western blot analyses. Correlation analysis was performed between the miRNAs and target proteins. Results: The relative content of miR-20a and miR-20b was lower in the center of the tumor than at the edge in the LIMG, lower at the edge of the tumor than in the center in the HIMG, and lower in breast cancer tissues than in normal tissues. VEGF-A and HIF-1alpha mRNA levels were higher in the HIMG than in the LIMG, and levels were higher in both groups than in the normal group; there was no difference in mRNA levels between the edge and center of the tumor. VEGF-A and HIF-1alpha protein levels were higher in the HIMG than in the LIMG, and protein levels in both groups were higher than in the normal group; there was a significant difference in protein expression between the edge and center of the tumor. Correlation analysis showed that the key miRNAs (miR-20a and miR-20b) negatively correlated with the target proteins (VEGF-A and HIF-1alpha). Conclusions: Our data suggest that miR-20a and miR-20b are differentially distributed in breast cancer, while VEGF-A and HIF-1alpha mRNA had coincident distributions, and VEGF-A and HIF-1alpha proteins had uneven and opposing distributions to the miRNAs. It appears that one of the most important facets underlying metastatic heterogeneity is the differential distribution of miR-20a and miR-20b and their regulation of target proteins.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.2
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• pp.871-876
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• 2014

Objective: Desmogleins (DSGs) are major members among the desmosomal cadherins critically involved in cell-cell adhesion and the maintenance of normal tissue architecture in epithelia. Reports exploring links of DSG family member expression with cancers are few and vary. The aim of this study was to investigate the ratio of DSG2 and DSG3 mRNA expression in esophageal squamous cell carcinoma (ESCC) tissue to normal tissue (T/N ratio) and evaluate correlations with clinical parameters. Methods: The mRNA expression of DSGs, as well as ${\gamma}$-catenin and desmoplakin, was detected by real-time quantitative RT-PCR in 85 cases of ESCC tissue specimens. Results: The expression level of DSG3 mRNA was significantly higher than that of DSG2 in ESCC specimens (p=0.000). DSG3 mRNA expression highly correlated with histological grade (p=0.009), whereas that of DSG2 did not significantly relate to any clinicopathologic parameter. Kaplan-Meier survival analysis showed that only DSG3 expression had an impact on the survival curve, with negative DSG3 expression indicating worse survival (p=0.038). Multivariate Cox regression analysis demonstrated DSG3 to be an independent prognostic factor for survival. Furthermore, correlation analysis demonstrated the mRNA level of DSG3 to highly correlate with those of ${\gamma}$-catenin and desmoplakin in ESCC samples (p=0.000), implying that the expression of desmosomal components might be regulated by the same upstream regulatory molecules. Conclusions: Our findings suggest that DSG3 may be involved in the progression of ESCC and serve as a prognostic marker, while expression of DSG2 cannot be used as a predictor of ESCC patient outcome.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3431-3436
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• 2016

Background: Carcinogenesis is a multifaceted intricate cellular mechanism of transformation of the normal functions of a cell into neoplastic alterations. Metastasis may result in failure of conventional treatment and death Hence, research on metastatic suppressors in cancer is a high priority. The metastatic suppressor gene CD82, also known as KAI1, is a member of the transmembrane 4 superfamily which was first identified in carcinoma of prostate. Little work has been done on this gene in breast cancer. Herein, we aimed to determine the gene and protein level expression of CD82/KAI1 in breast cancer and its role as a prognosticator. Materials and Methods: In this study, 83 histologically proven cases of breast cancer and a similar number of controls were included. Patient age ranged from 18-70 years. Quantitative Real Time Polymerase Chain Reaction (q-RT PCR) and immunohistochemistry (IHC) were used to investigate KAI1 expression at gene and protein levels, respectively. Statistical analysis was done to correlate expression of KAI1 and clinicopathological parameters. Results: It was revealed that: (i) KAI1 was remarkably diminished in metastatic vs non metastatic breast cancer both at the gene and the protein levels (P < .05); (ii) KAI1 expression levels were strongly correlated with TNM staging, histological grade and advanced stage (p<0.001) and no association was found with any other studied parameter; (iii) Lastly, a significant correlation was observed between expression of KAI1 and overall median survival of BC patients (P = 0.04). Conclusions: Our results suggest that lack of expression of the KAI1 might indicate a more aggressive form of breast cancer. Loss of KAI1 may be considered a significant prognostic marker in predicting metastatic manifestation. When evaluated along with the clinical and pathological factors, KAI1 expression may be beneficial to tailor aggressive therapeutic strategies for such patients.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.7
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• pp.3213-3222
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• 2016

Background: Methyl donor status influences DNA stability and DNA methylation although little is known about effects on DNA methyltransferases. The aim of this study was to determine whether methyl-donor status influences DNA methyltransferase (Dnmt) gene expression in cervical cancer cells, and if so, whether there are associated effects on global DNA methylation. Materials and Methods: The human cervical cancer cell line, C4-II, was grown in complete medium and medium depleted of folate (F-M+) and folate and methionine (F-M-). Growth rate, intracellular folate, intracellular methionine and homocysteine in the extracellular medium were measured to validate the cancer cell model of methyl donor depletion. Dnmt expression was measured by qRT-PCR using relative quantification and global DNA methylation was measured using a flow cytometric method. Results: Intracellular folate and methionine concentrations were significantly reduced after growth in depleted media. Growth rate was also reduced in response to methyl donor depletion. Extracellular homocysteine was raised compared with controls, indicating disturbance to the methyl cycle. Combined folate and methionine depletion led to a significant down-regulation of Dnmt3a and Dnmt3b; this was associated with an 18% reduction in global DNA methylation compared with controls. Effects of folate and methionine depletion on Dnmt3a and 3b expression were reversed by transferring depleted cells to complete medium. Conclusions: Methyl donor status can evidently influence expression of Dnmts in cervical cancer cells, which is associated with DNA global hypomethylation. Effects on Dnmt expression are reversible, suggesting reversible modulating effects of dietary methyl donor intake on gene expression, which may be relevant for cancer progression.

• Fisheries and Aquatic Sciences
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• v.19 no.10
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• pp.40.1-40.14
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• 2016

Background: Due to the paucity of oceanic resources utilized in the preparation of diets for cultured fish, commercial feed producers have been trying to replace fishmeal (FM) using alternative protein sources such as vegetable protein meals (VMs). One of the main drawbacks of using VMs in fish feed is related to the presence of a variety of anti-nutritional factors, which could trigger an inflammation process in the distal intestine. This reduces the capacity of the enterocytes to absorb nutrients leading to reduced fish growth performances. Methods: We evaluated the mitigating effects of butyrate and taurine used as feed additives on the morphological abnormalities caused by a soybean meal (SBM)-based diet in the distal intestine of sea bass (Dicentrarchus labrax). We used three experimental diets, containing the same low percentage of FM and high percentage of SBM; two diets were supplemented with either 0.2% sodium butyrate or taurine. Histological changes in the intestine of fish were determined by light and transmission electron microscopy. Infiltration of $CD45^+$ leucocytes in the lamina propria and in the submucosa was assessed by immunohistochemistry. We also quantified by One-Step Taqman$^{(R)}$ real-time RT-PCR the messenger RNA (mRNA) abundance of a panel of genes involved in the intestinal mucosa inflammatory response such as $TNF{\alpha}$ (tumor necrosis factor alpha) and interleukins: IL-8, IL-$1{\beta}$, IL-10, and IL-6. Results: Fish that received for 2 months the diet with 30% soy protein (16.7% SBM and 12.8% full-fat soy) developed an inflammation in the distal intestine, as confirmed by histological and immunohistochemistry data. The expression of target genes in the intestine was deeply influenced by the type of fish diet. Fish fed with taurine-supplemented diet displayed the lowest number of mRNA copies of IL-$1{\beta}$, IL-8, and IL-10 genes in comparison to fish fed with control or butyrate-supplemented diets. Dietary butyrate caused an upregulation of the $TNF{\alpha}$ gene transcription. Among the quantified interleukins, IL-6 was the only one to be not influenced by the diet. Conclusions: Histological and gene expression data suggest that butyrate and taurine could have a role in normalizing the intestinal abnormalities caused by the SBM, but the underling mechanisms of action seem different.

• Herbal Formula Science
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• v.25 no.1
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• pp.1-10
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• 2017

Objectives : The fruit of Prunus mume Siebold & Zucc. has been used as an alternative medicine and functional food in Korea and Japan for preventive and therapeutic purposes. However, its molecular actions and mechanism on anti-inflammatory activity have not been clearly investigated. The aim of this study was to clarify the anti-inflammatory activity of the ethanol extract of P. mume fruit (EEPM) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and sought to understand the associated molecular mechanisms. Methods : Cytotoxicity was assessed by an MTT assay. The amount of nitric oxide (NO) production was determined by nitrite assay. The mRNA expression of inducible nitric oxide synthase (iNOS) was analyzed by RT-PCR. In addition, expression levels of iNOS, nuclear factor-erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) protein were detected by Western blotting. Results : Our data indicated that EEPM inhibited NO production in LPS-stimulated RAW264.7 cells in a concentration-dependent manner. At the mRNA and protein levels, EEPM suppressed LPS-induced iNOS expression. On the other hand, EEPM markedly enhanced HO-1 expression, which was associated with an induction and nuclear translocation of Nrf2. Moreover, the inhibitory effect of EEPM against LPS‑induced NO production was significantly enhanced by hemin, a HO-1 inducer; however, EEPM's effect on the production of NO was abolished by zinc protoporphyrin IX, a HO-1 inhibitor. Conclusion : The results suggest that EEPM can act as a suppressor agent on NO production through an activation of Nrf2/HO-1 signaling pathway, and may be a promising candidate for the treatment of inflammatory diseases.